Molecular determinants of protein evolvability.

The plethora of biological functions that sustain life is rooted in the remarkable evolvability of proteins. An emerging view highlights the importance of a protein's initial state in dictating evolutionary success. A deeper comprehension of the mechanisms that govern the evolvability of these initial states can provide invaluable insights into protein evolution. In this review, we describe several molecular determinants of protein evolvability, unveiled by experimental evolution and ancestral sequence reconstruction studies. We further discuss how genetic variation and epistasis can promote or constrain functional innovation and suggest putative underlying mechanisms. By establishing a clear framework for these determinants, we provide potential indicators enabling the forecast of suitable evolutionary starting points and delineate molecular mechanisms in need of deeper exploration.

Discovery and Characterisation of Highly Cooperative FAK-Degrading PROTACs.

Focal adhesion kinase (FAK) is a key mediator of tumour progression and metastasis. To date, clinical trials of FAK inhibitors have reported disappointing efficacy for oncology indications. We report the design and characterisation of GSK215, a potent, selective, FAK-degrading Proteolysis Targeting Chimera (PROTAC) based on a binder for the VHL E3 ligase and the known FAK inhibitor VS-4718. X-ray crystallography revealed the molecular basis of the highly cooperative FAK-GSK215-VHL ternary complex, and GSK215 showed differentiated in-vitro pharmacology compared to VS-4718. In mice, a single dose of GSK215 induced rapid and prolonged FAK degradation, giving a long-lasting effect on FAK levels (≈96 h) and a marked PK/PD disconnect. This tool PROTAC molecule is expected to be useful for the study of FAK-degradation biology in vivo, and our results indicate that FAK degradation may be a differentiated clinical strategy versus FAK inhibition for the treatment of cancer.

Epistasis arises from shifting the rate-limiting step during enzyme evolution of a ß-lactamase.

Epistasis, the non-additive effect of mutations, can provide combinatorial improvements to enzyme activity that substantially exceed the gains from individual mutations. Yet the molecular mechanisms of epistasis remain elusive, undermining our ability to predict pathogen evolution and engineer biocatalysts. Here we reveal how directed evolution of a ß-lactamase yielded highly epistatic activity enhancements. Evolution selected four mutations that increase antibiotic resistance 40-fold, despite their marginal individual effects (≤2-fold). Synergistic improvements coincided with the introduction of super-stochiometric burst kinetics, indicating that epistasis is rooted in the enzyme's conformational dynamics. Our analysis reveals that epistasis stemmed from distinct effects of each mutation on the catalytic cycle. The initial mutation increased protein flexibility and accelerated substrate binding, which is rate-limiting in the wild-type enzyme. Subsequent mutations predominantly boosted the chemical steps by fine-tuning substrate interactions. Our work identifies an overlooked cause for epistasis: changing the rate-limiting step can result in substantial synergy that boosts enzyme activity.

Pervasive epistasis exposes intramolecular networks in adaptive enzyme evolution.

Enzyme evolution is characterized by constant alterations of the intramolecular residue networks supporting their functions. The rewiring of these network interactions can give rise to epistasis. As mutations accumulate, the epistasis observed across diverse genotypes may appear idiosyncratic, that is, exhibit unique effects in different genetic backgrounds. Here, we unveil a quantitative picture of the prevalence and patterns of epistasis in enzyme evolution by analyzing 41 fitness landscapes generated from seven enzymes. We show that >94% of all mutational and epistatic effects appear highly idiosyncratic, which greatly distorted the functional prediction of the evolved enzymes. By examining seemingly idiosyncratic changes in epistasis along adaptive trajectories, we expose several instances of higher-order, intramolecular rewiring. Using complementary structural data, we outline putative molecular mechanisms explaining higher-order epistasis along two enzyme trajectories. Our work emphasizes the prevalence of epistasis and provides an approach to exploring this phenomenon through a molecular lens.

Epistasis and intramolecular networks in protein evolution.

Proteins are molecular machines composed of complex, highly connected amino acid networks. Their functional optimization requires the reorganization of these intramolecular networks by evolution. In this review, we discuss the mechanisms by which epistasis, that is, the dependence of the effect of a mutation on the genetic background, rewires intramolecular interactions to alter protein function. Deciphering the biophysical basis of epistasis is crucial to our understanding of evolutionary dynamics and the elucidation of sequence-structure-function relationships. We featured recent studies that provide insights into the molecular mechanisms giving rise to epistasis, particularly at the structural level. These studies illustrate the convoluted and fascinating nature of the intramolecular networks co-opted by epistasis during the evolution of protein function.